Figure 3
Knockdown of SMAD1/5/8 impairs Notch-induced chondrocytes differentiation.
ATDC5 cells were transfected with SMAD1/5/8 siRNA oligos or NICD1 plasmids for 48 hours before being harvested for western blot and RT-PCR analysis. (A) A drastic reduction in SMAD1/5/8 protein expression was seen in cells that had SMAD1/5/8 knocked down. (B) SMAD1/5/8 knocking down significantly inhibited AP, Runx2, MMP13, p57 gene expression and resulted in the reduction of NICD1-induced gene expression. (C) Immunoprecipitation (IP) by p-SMAD1/5/8 antibody showed an interaction between p-SMAD1/5/8 and NICD1. (D) Immunoprecipitation by anti-NICD1 antibody showed a strong interaction between SMAD1/5/8 and NICD1. Normal lgG serving as negative control. Data B are means ± SD of three independent experiments performed in duplicate and the control gene expression level was set at 1. (*p < 0.05 compared with control groups; #p < 0.05 compared with NICD1 alone cells).
